1. The periciliary membrane complex, Tubby family of proteins and ciliary trafficking of GPCR Tubby is the founding member of the Tubby-like family of proteins. The naturally occurring tubby mutation in mice causes retinitis pigmentosa, hearing loss and obesity. We show that the tubby is required for the correct localization of a select group of membrane receptors to the distal portion of primary and sensory cilia. By using tubby and tubby like protein 1 as baits, we have identified interacting proteins ankrd54 and an unknown protein LOC69239. Preliminary studies indicate that these proteins are likely to function in ciliary trafficking and we have engineered knockout mouse lines that carry mutations in these genes. Phenotype analysis is underway. We further hypothesize that phosphatidyl inoside bisphosphate (PIP2) is likely to be a critical signaling molecule for ciliary trafficking and experiments are underway to delineate the mechanism of this transport process. 2. Macf1 is required for microtubule and actin interactions during ciliated sensory cell maturation and function. The cytoskeleton, present in all eukaryotic cells, is comprised of microtubules, actin filaments and intermediate filaments, and forms important structures such as cilia and lamellipodia. It plays vital roles during cell division and intracellular transport. Spectraplakins are cytoskeletal cross-linkers, which associate with F-actin, microtubules and intermediate filaments to integrate cytoskeletal networks. In vertebrates, in addition to their role as cytoskeletal cross-linkers, spectraplakins have also been shown to play a role in intracellular trafficking. Microtubule actin cross linking factor 1 (MACF1/ACF7), one of only two spectraplakin proteins found in mammalian systems, is a 586 kDa protein, highly conserved across species. Previous work has shown that Macf1 plays a role in integrating coordinated microtubule and actin dynamics along cell borders (focal adhesions/adherens junctions). In mouse, Macf1 was found to be the third most abundant protein peptide in photoreceptor sensory cilium proteome and the Drosophila homolog shot, is to thought act as an actin-MT cross-linker during photoreceptor morphogenesis. We identified that Macf1 interacts with centrosomal/basal body protein Mkks and therefore may contribute to ciliogenesis. We generated conditional knock out mice, in which Macf1 had been deleted in developing ciliated sensory tissues (retina, cochlea, lateral ventricles) and Macf1 antibodies against its actin-binding domain. Immunohistochemistry suggests that Macf1 is widely expressed in developing retina, being concentrated at the apical border of the neuroepithelium. Loss of Macf1 in the developing retina results in a complete loss of visual function due to severely disrupted retinal lamination primarily affecting photoreceptors. Loss of apical basal polarity of the outer nuclear layer is seen as early as P5, which is preceded by ectopic ciliary vesicles and loss of basal body migration and/or docking at the apical membrane. Integrity of junctional markers (adherens junctions and tight junctions) is not compromised but polarity components RhoA and Par3 are differentially expressed. In developing cochlea sensory neurons, Macf1 expression is highly enriched at the base of the kinocilium. Mutant cochlea sensory neurons display mild polarity defects yet the cochlea length and tissue organization is not disrupted, suggesting that basal body migration is one of the underlying defects. Development of ciliated ventricular neuroepithelia is dependent on the correct polarization of primary cilia. Loss of Macf1 severely hinders the development of the lateral ventricle. These findings suggest a critical role for Macf1 in developing sensory neurons, and highlight possible novel functions for microtubule and actin interactions in ciliary related processes. These insights provide the cellular basis for clinical phenotypes associated with ciliopathies. We are currently investigating whether Macf1 also plays a separate role in mature photoreceptors at the point of actin-microtubule network connection for proteins en route to the outer segments via the connecting cilia. 3. Planar cell polarity (PCP) and retinal development Planar cell polarity (PCP) signaling plays a critical role in tissue morphogenesis. In mammals, disruption of three of the six core PCP components results in polarity-dependent defects with rotated cochlear hair cell stereocilia and open neural tube. We recently demonstrated a role of Prickle1, a core PCP molecule in Drosophila, in mammalian neuronal development. To examine Prickle1 function along a broader developmental window, we generated three mutant alleles in mice. We show that the complete loss of Prickle1 leads to systemic tissue outgrowth defects, aberrant cell organization and disruption of polarity machinery. Prickle1 mutants recapitulate the characteristic features of human Robinow syndrome and phenocopy mouse mutants with Wnt5a or Ror2 gene defects, prompting us to explore an association of Prickle1 with the Wnt pathway. We show that Prickle1 is a proteasomal target of Wnt5a signaling and that Dvl2, a target of Wnt5a signaling, is misregulated in Prickle1 mutants. Our studies implicate Prickle1 as a key component of the Wnt-signaling pathway and suggest that Prickle1 mediates some of the WNT5A-associated genetic defects in Robinow syndrome. Current ongoing studies examine the role of Prickle 1 and Prickle 2 in retinal development and homeostasis. 4. Gene therapy for retinal degeneration Tubby-like protein 1 (TULP1) is a photoreceptor specific protein expressed in both rods and cones. Gene defects in Tulp1 cause retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) in humans. A knockout mouse model lacking Tulp1 (TULP1/) develops rapid photoreceptor degeneration with severe mislocalization of rod and cone opsins. The human and mouse Tulp1 genes are poorly conserved and there is an additional exon in the human gene that is not represented in the mouse ortholog. Curiously, the human TULP1 gene proved unclonable. We therefore generated a synthetic gene that substantially altered the nucleotide sequence without changing the amino acid codons. We validated the efficacy of the synthetic human TULP1 replacement gene controlled by a promoter derived from the human rhodopsin kinase (RK) gene which is active in both rods and cones. We found substantial rescue of the disease phenotype as a result of transgene expression. This is another gene therapy study in which both rods and cones were targeted successfully with a single photoreceptor-specific promoter. This work is being published.